Intramural Nerve Fibers Research Articles

Role of PTHrP in attenuating transient pressure rises and associated afferent nerve activity of the rat bladder.

Parathyroid hormone-related protein (PTHrP) released from detrusor smooth muscle (DSM) as the bladder fills acts as an endogenous DSM relaxant to facilitate bladder storage function. Here, the effects of exogenous PTHrP on transient pressure rises (TPRs) in the bladder and associated afferent nerve activity during bladder filling were investigated. In anaesthetized rats, changes in the intravesical pressure were measured while the bladder was gradually filled with saline. Afferent nerve activity was simultaneously recorded from their centrally disconnected left pelvic nerves. In DSM strips, spontaneous and nerve-evoked contractions were isometrically recorded. The distribution of PTHrP receptors (PTHrPRs) in the bladder wall was also examined by fluorescence immunostaining. The bladders in which the contralateral pelvic nerve was also centrally disconnected developed nifedipine, an L-type voltage-dependent Ca2+ channel blocker-sensitive TPRs (< 3mmHg). Intravenous administration of PTHrP suppressed these TPRs and associated bursts of afferent nerve activity. In the bladders with centrally connected contralateral pelvic nerves, atropine, a muscarinic receptor antagonist-sensitive large TPRs (> 3mmHg) developed in the late filling phase. PTHrP diminished the large TPRs and corresponding surges of afferent nerve activity. In DSM strips, bath-applied PTHrP (10nM) suppressed spontaneous phasic contractions, while less affecting nerve-evoked contractions. PTHrPRs were expressed in DSM cells but not in intramural nerve fibers. Thus, PTHrP appears to suppress bladder TPRs and associated afferent nerve activity even under the influence of low degree of parasympathetic neural input during storage phases. Endogenous PTHrP may indirectly attenuate afferent nerve activity by suppressing TPRs to facilitate urinary accommodation.

The Influence of an Adrenergic Antagonist Guanethidine on the Distribution Pattern and Chemical Coding of Caudal Mesenteric Ganglion Perikarya and Their Axons Supplying the Porcine Bladder.

This study was aimed at disclosing the influence of intravesically instilled guanethidine (GUA) on the distribution, relative frequency and chemical coding of both the urinary bladder intramural sympathetic nerve fibers and their parent cell bodies in the caudal mesenteric ganglion (CaMG) in juvenile female pigs. GUA instillation led to a profound decrease in the number of perivascular nerve terminals. Furthermore, the chemical profile of the perivascular innervation within the treated bladder also distinctly changed, as most of axons became somatostatin-immunoreactive (SOM-IR), while in the control animals they were found to be neuropeptide Y (NPY)-positive. Intravesical treatment with GUA led not only to a significant decrease in the number of bladder-projecting tyrosine hydroxylase (TH) CaMG somata (94.3 ± 1.8% vs. 73.3 ± 1.4%; control vs. GUA-treated pigs), but simultaneously resulted in the rearrangement of their co-transmitters repertoire, causing a distinct decrease in the number of TH+/NPY+ (89.6 ± 0.7% vs. 27.8 ± 0.9%) cell bodies and an increase in the number of SOM-(3.6 ± 0.4% vs. 68.7 ± 1.9%), calbindin-(CB; 2.06 ± 0.2% vs. 9.1 ± 1.2%) or galanin-containing (GAL; 1.6 ± 0.3% vs. 28.2 ± 1.3%) somata. The present study provides evidence that GUA significantly modifies the sympathetic innervation of the porcine urinary bladder wall, and thus may be considered a potential tool for studying the plasticity of this subdivision of the bladder innervation.

The Neurochemical Characterization of Parasympathetic Nerve Fibers in the Porcine Uterine Wall Under Physiological Conditions and After Exposure to Bisphenol A (BPA)

Bisphenol A, a substance commonly used in plastic manufacturing, is relatively well known as an endocrine disruptor, which may bind to estrogen receptors and has multidirectional negative effects on both human and animal organisms. Previous studies have reported that BPA may act on the reproductive organs, but knowledge concerning BPA-induced changes within the nerves located in the uterine wall is extremely scant. The aim of this study was to investigate the impact of various doses of BPA on the parasympathetic nerves located in the corpus and horns of the uterus using a single and double immunofluorescence method. The obtained results have shown that BPA may change not only the expression of vesicular acetylcholine transporter (VAChT—a marker of parasympathetic nervous structures) in the uterine intramural nerve fibers, but also the degree of colocalization of this substance with other neuronal factors, including substance P (SP), vasoactive intestinal polypeptide (VIP), galanin (GAL), and calcitonin gene–related peptide (CGRP). Moreover, BPA caused changes in the density of the overall populations of fibers immunoreactive to the particular neuropeptides mentioned above. The characteristics of the changes clearly depended on the part of the uterus, the neuronal factors studied, and the dosage of BPA. The mechanisms of the observed fluctuations are probably connected with the neurotoxic and/or pro-inflammatory activity of BPA. Moreover, the results have shown that even low doses of BPA are not neutral to living organisms. Changes in the neurochemical characterization of nerves supplying the uterine wall may be the first subclinical sign of intoxication with this substance.

Neurochemical characterization of intramural nerve fibres in the porcine oesophagus.

The gastrointestinal (GI) tract is innervated by nerve processes derived from the intramural enteric neurons and neurons localized outside the digestive tract. This study analysed the neurochemical characterization of nerves in the wall of the porcine oesophagus using single immunofluorescence technique. Immunoreactivity to vesicular acetylcholine transporter (VAChT), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), somatostatin (SOM), galanin (GAL), neuronal isoform of nitric oxide synthase (nNOS), substance P (SP), leucine enkephalin (LENK), calcitonin gene-related peptide (CGRP) or dopamine beta-hydroxylase (DBH) was investigated in intramuscular and intramucosal nerves of the cervical, thoracic and abdominal oesophagus. The results indicate that all of the substances studied were present in the oesophageal nerves. The density of particular populations of fibres depended on the segment of the oesophagus. The most numerous were fibres immunoreactive to VIP in the longitudinal and circular muscle layers of the abdominal oesophagus: The number of these fibres amounted to 16.4±0.8 and 18.1±3.1, respectively. In turn, the least numerous were CGRP-positive fibres, which were present only in the circular muscle layer of the cervical oesophagus and mucosal layer of the abdominal oesophagus in the number of 0.3±0. The obtained results show that nerves in the porcine oesophageal wall are very diverse in their neurochemical coding, and differences between particular parts of the oesophagus suggest that organization of the innervation clearly depends on the fragment of this organ.

Influence of bisfenol A on chemical coding of the nerve fibers of the cardiac apex in the domestic pig

The study was designed to determine the effects of bisphenol A (BPA) on chemical coding of intramural nerve fibers localized in the cardiac apex of domestic pigs. Investigations were performed on 10 immature gilts. The animals were divided into two groups: control (Group K) and experimental (Group B). Gilts from Group B received capsules containing bisphenol A (0.5 mg / kg m.c / day) for 28 days. After euthanasia, fragments of the cardiac apex were collected and subjected to the standard single immunofluorescence method, using primary antisera directed against neuropeptide Y (NPY), vesicular transporter acetylcholine (VAChT), tyrosine hydroxylase (TH), substance P (SP), transcript regulated cocaine and amphetamine peptide (CART) and gene calcitonin (CGRP), as well as secondary antibodies conjugated with fluorochromes: Alexa fluor 488 or Alexa fluor 546. The present studies demonstrated that orally administered bisphenol A causes a marked increase in the number of nerve fibers immunoreactive to NPY, VAChT, TH and SP. On the other hand, CART and CGRP immunoreactive fibers have not been detected during the present study either in healthy animals or in pigs after BPA administration. Bisphenol A affects the neurochemical profile of nerves located in the cardiac apex of domestic pigs. These fluctuations in the chemical coding of nerve fibers may be associated with neuroprotective and adaptive processes in the myocardium. However, as of yet the mechanisms of these changes are not fully explained. They may be connected with the direct action of BPA on the myocardium and/or general neurodegenerative effects of this substance on the nervous system.

Platelet‐derived growth factor receptor‐α cells in mouse urinary bladder: a new class of interstitial cells

Specific classes of interstitial cells exist in visceral organs and have been implicated in several physiological functions including pacemaking and mediators in neurotransmission. In the bladder, Kit+ interstitial cells have been reported to exist and have been suggested to be neuromodulators. More recently a second interstitial cell, which is identified using antibodies against platelet-derived growth factor receptor-α (PDGFR-α) has been described in the gastrointestinal (GI) tract and has been implicated in enteric motor neurotransmission. In this study, we examined the distribution of PDGFR-α+ cells in the murine urinary bladder and the relation that these cells may have with nerve fibres and smooth muscle cells. Platelet-derived growth factor receptor-α+ cells had a spindle shape or stellate morphology and often possessed multiple processes that contacted one another forming a loose network. These cells were distributed throughout the bladder wall, being present in the lamina propria as well as throughout the muscularis of the detrusor. These cells surrounded and were located between smooth muscle bundles and often came into close morphological association with intramural nerve fibres. These data describe a new class of interstitial cells that express a specific receptor within the bladder wall and provide morphological evidence for a possible neuromodulatory role in bladder function.

The simultaneous presence of neuroepithelial cells and neuroepithelial bodies in the respiratory gas bladder of the longnose gar, Lepisosteus osseus, and the spotted gar, L. oculatus

Anatomical and functional studies on the autonomic innervation as well as the location of airway receptors in the air-bladder of lepisosteids are very fragmentary. These water-breathing fishes share in common with the bichirs the presence of a glottis (not a ductus pneumaticus) opening into the esophagus. In contrast to a high concentration of neuroepithelial cells (NECs) contained in the furrowed epithelium in the lung of Polypterus, these cells are scattered as solitary cells in the glottal epithelium, and grouped to form neuroepithelial bodies (NEBs) in the mucociliated epithelium investing the main trabeculae in the air-bladder of Lepisosteus osseus and L. oculatus. The present immunohistochemical studies also demonstrated the presence of nerve fibers in the trabecular striated musculature and a possible relation to NEBs in these species, and identified immunoreactive elements of this innervation. Tyrosine hydroxylase (TH), choline acetyltransferase (ChAT), 5-HT and neuropeptide immunoreactivities were detected in the intramural nerve fibers. 5-HT and VIP immunopositive nerve fibers are apparently associated with NEBs. TH, VIP and SP immunoreactivities are also present in nerve fibers coursing in the radially arranged striated muscle surrounding the glottis and its submucosa. 5-HT positive neurons are also found in submucosal and the muscle layers of the glottis. The physiological function of the adrenergic and inhibitory innervation of the striated muscle as well as the neurochemical coding and morphology of the innervation of the NEBs are not known. Future studies are needed to provide evidence for these receptors with the capacity of chemoreceptors and/or mechanoreceptors.

Endo- and exocytosis of vesicles in the intramural nerve fibers of the rat right atrium

Endo- and exocytosis of vesicles in the intramural nerve fibers of the rat right atrium

Effects of Exogenous Glutamate and Kainate on Electric Field-stimulated Contractions of Isolated Human Ureter

A neurotransmitter role for glutamate in the autonomous nervous system was recently demonstrated in the gastrointestinal tract, and its stimulatory effect on spontaneous motility of human ureter was shown. The aim of our study was to investigate the effects of glutamate on the release of neurotransmitters from intramural nerves of the human ureter. The effects of exogenous glutamate were tested on electric field-stimulated contractions of isolated human ureter, taken from 16 adult patients after nephrectomy. The longitudinal tension and intraluminal pressure of the isolated ureter were recorded simultaneously. The electric field stimulation was done with square wave pulses (20 V through electrodes, 400 mA, duration 1 ms, frequency 16 Hz). The pulse trains lasted for 30 s, a with 30-s pause. Glutamate (7.9 x 10(-6) M/L to 10.6 x 10(-3) M/L) and kainic acid (6.3 x 10(-8) M/L to 2.2 x 10(-5) M/L) produced a concentration-dependent decrease in the electric field-stimulated activity of the isolated preparations. However, N-methyl-D-aspartic acid (9.1 x 10(-8) M/L to 3.1 x 10(-5) M/L), (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (7.2 x 10(-8) M/L to 3.2 x 10(-6) M/L) and (+/-)-1-Aminocyclopentane-trans-1,3-dicarboxylic acid (7.7 x 10(-8) M/L to 6.5 x 10(-5) M/L) were ineffective. The electric field-stimulated contractions of isolated ureter were also inhibited by lidocaine (3.70 x 10(-4)M/L) and atropine (1.00 x 10(-6)M/L). The results of our study suggest that glutamate inhibits electric field-stimulated release of acetylcholine in the human ureter through activation of kainate ionotropic receptors, located on the intramural nerve fibers.

Innervation and Neurotransmitter Localization in the Lung of the Nile bichir Polypterus bichir bichir

Anatomical and functional studies of the autonomic innervation in the lung of dipnoan fishes and the bichirs are lacking. The present immunohistochemical studies demonstrated the presence of nerve fibers in the muscle layers of the lung of the bichir, Polypterus bichir bichir, and identified the immunoreactive elements of this innervation. Tyrosine hydroxylase, acetylcholinesterase, and peptide immunoreactivity was detected in the intramural nerve fibers. Extensive innervation was present in the submucosa where adenylatecyclase/activating polypeptide 38, substance P, P(2)X(2), and 5-hydroxytryptamine (5-HT)-immunoreactive nerve fibers mainly supplied blood vessels. A collection of monopolar neurons located in the submucosal and the muscular layers of the glottis expressed a variety of various transmitters. These neurons may be homologous to ganglion cells in the branchial and pharyngeal rami of the vagus in fishes. Nerves containing 5-HT and P(2)X(2) receptor immunoreactivity projected to the lung epithelium. Associated with neuroepithelial cells in mucociliated epithelium, were neuronal nitric oxide synthase-immunopositive axons. The physiological function of this innervation is not known. The present study shows that the pattern of autonomic innervation of the bichir lung may by similar in its elements to that in tetrapods.

Effect of exogenous glutamate and N‐Methyl‐D‐aspartic acid on spontaneous activity of isolated human ureter

While the neurotransmitter role of glutamate in the gastrointestinal tract has been shown, its effects on smooth muscle of the human ureter have not previously been investigated. In our study we have investigated the effects of exogenous glutamate on the spontaneous activity of isolated human ureter, taken from 14 adult patients after nephrectomy. The segment of ureter, excised 3 cm distal from the pyeloureteral junction, was isolated in an organ bath. Both longitudinal tension and intraluminal pressure of the segment were recorded simultaneously. Glutamate administered in the lumen of the isolated ureteral segments (7.8 x 10(-7) M/L-3.5 x 10(-2) M/L) was ineffective. When added to the isolated organ bath from the serous side of the ureteral segment, glutamate (7.9 x 10(-6) M/L-10.6 x 10(-3) M/L) and N-Methyl-D-aspartic acid (NMDA) (9.1 x 10(-8) M/L-3.1 x 10(-5) M/L) produced a concentration-dependent increase in spontaneous activity of the isolated preparations, while kainic acid (6.3 x 10(-8) M/L-10.5 x 10(-5) M/L) and (+/-)-trans-1-Aminocyclopentane-trans-1,3-dicarboxylic acid (ACPD) (7.7 x 10(-8) M/L -6.5 x 10(-5) M/L) were ineffective. The results of our study suggest that an excitatory neurotransmitter glutamate stimulates spontaneous activity of the human ureter through activation of NMDA ionotropic receptors, located on smooth muscle cells or intramural nerve fibers.

Colocalisation of neuropeptides, nitric oxide synthase and immunomarkers for catecholamines in nerve fibres of the adult human vas deferens.

Single and double-label immunofluorescence methods were used to determine the distribution and patterns of colocalisation of various neuropeptides and nitric oxide synthase (NOS) with the catecholamine synthesising enzymes tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DbetaH) in nerve fibres within specimens of adult human vas deferens obtained at vasectomy (age range 28 to 83 y). Cholinergic nerve fibres were immunolabelled with an antiserum to vesicular acetylcholine transporter (VAChT). Using the general nerve marker protein gene product 9.5 (PGP) the density of intramural nerve fibres was found to be similar irrespective of age. Many of these axons, especially in the outer 2 muscle layers were TH and DbetaH-immunoreactive (IR) and were thus confirmed as noradrenergic. Fewer such axons were seen in the inner longitudinal muscle layer. All the noradrenergic nerve fibres also displayed NPY-immunoreactivity with minor populations containing galanin (GAL) or somatostatin (SOM). Nerve fibres lacking TH and DbetaH-IR were immunoreactive for VAChT and were sparsely distributed throughout the 2 outer muscle layers but more numerous in the inner muscle layer. Nerves lacking TH and DbetaH were immunoreactive for NPY and some also contained NOS, VIP or CGRP. These results have been compared with those obtained previously from specimens of human neonatal and infant vas deferens where, in contrast to the present results, NOS and VIP were shown to be colocalised with TH in many of the intramuscular nerve fibres. It thus appears that NOS and VIP cease their coexistence with TH in intramuscular nerve fibres of the human vas deferens between the pre- and postpubertal states. In addition to the intramuscular nerve fibres a VAChT-IR subepithelial nerve plexus occurs in the vas deferens and may control the secretory activity of the lining epithelium. Most of these subepithelial nerve fibres were immunoreactive for NPY and many also contained VIP while minor populations were immunoreactive for NOS, GAL, SOM or SP although fibres containing CGRP were not observed. The neuropeptide content of the subepithelial nerve plexus was similar to that observed in the infant, except for an increased density of VIP-IR nerves, which may reflect greater activity of the lining epithelial cells in the adult vas deferens.

Morphological Investigations of the Forestomach in Cows Suffering From Hoflund Syndrome

Since HOFLUND's explanation (1) of a functional stenosis of either the bovine forestomach or the pylorus due to damages of the vagal nerve in 1940, this hypothesis is discussed controversially. In addition disturbances of the beforementioned extrinsic nerve system, mechanical influences are discussed to be the origin of this disease (2,3). As an extrinsic control system the vagal nerves are responsible for a coordionated contraction of the ruminant forestomach. These nerves originating from the gastric centre in the medulla oblongata are consisting of predominantly afferent fibres and some efferent, preganglionic nerve fibres ( 4). The efferent fibres are communicating with intramurally located ganglia. Recently published data present that the intramural neurons, also named as intrinsic nerve system are able to initiate reticulo-ruminal contraction (9,10).&#x0D; Purpose of this study was to evaluate morphological alterations ofvagal and intramural nerve fibres, intramural neurons and inflamamtory changes in the wall of the forestomachs and abomasum of cows suffering from HOFLUND's Syndrome.

Effect of transmural vagal stimulation on electrotonic current spread in the rabbit sinoatrial node.

The effect of vagal stimulation on the decay of electrotonic potential caused by intracellular current injection and on input resistance was measured in the sinoatrial node of isolated rabbit right atria. Studies were performed on New Zealand White rabbits weighing approximately 2-3 kg. Vagal stimulation was achieved by transmural stimulation of intramural nerve fibres in the presence of propranolol. A K+ perfused suction electrode was used to inject hyperpolarising current pulses; input resistance was measured by means of a double barrel microelectrode. Vagal stimulation which caused a 14-20% increase of cycle length diminished electronic potential significantly by a decrease of membrane resistance. The input resistance of the sinoatrial node was not affected. Space constant values calculated by using either a one or a two dimensional model of electrotonic current spread were decreased on average by 13% and 14% respectively. The results from this study show that vagal stimulation which gave rise to a moderate negative chronotropic effect and marked changes in action potential configuration of nodal fibres affects the electrotonic interaction within the sinoatrial node. This may have consequences for the electrical activity and synchronisation of the sinoatrial nodal fibres.

Neurotransmission in human resistance arteries: contribution of alpha 1- and alpha 2-adrenoceptors but not P 2-purinoceptors.

The contribution of postjunctional alpha 1- and alpha 2-adrenoceptors and P2-purinoceptors to the neuroeffector response was examined in isolated segments of human subcutaneous resistance arteries. Electrical field stimulation (EFS, 20 V, 0.2 ms, 1-25 Hz) elicited a maximum contractile response which was 38.2 +/- 1.6% of that elicited by exogenously applied (5 microM) noradrenaline (n = 56). Tetrodotoxin (1 microM), used to inhibit neurotransmission, reduced the electrically evoked response to 24.7 +/- 4.4% (n = 10) of the control response. The alpha 1-adrenoceptor antagonist prazosin (1 microM) reduced the maximum EFS contractile response to 64.8 +/- 5.5% of the control response (n = 17). Application of the alpha 2-adrenoceptor antagonist yohimbine (0.1 microM) reduced the maximum EFS response to 68.2 +/- 8.2% of the control response (n = 9). In the presence of prazosin plus yohimbine at the above-mentioned concentrations the maximum response to EFS was reduced to 47.6 +/- 6.7% (n = 11). Responses following alpha-blockade were not statistically different from those in the presence of tetrodotoxin, but the mean responses indicate that a non-adrenergic component to the EFS response cannot be discounted. Desensitisation of P 2-purinoceptors with alpha, beta-methylene ATP had no effect on responses to EFS; therefore under the conditions studied these receptors do not appear to be involved in neurotransmission. These results confirm the presence of postjunctional alpha 1- and alpha 2-adrenoceptors in human resistance arteries and for the first time demonstrate that the postjunctional alpha 2-adrenoceptor is important in modulating vascular responses elicited by intramural sympathetic nerve fibres.

Inhibition by VIP and atriopeptin II on the field stimulation evoked release of [ 3H]noradrenaline in the rat portal vein

The modulatory effects of vasodilatory peptides on noradrenaline release from sympathetic nerve terminals have been studied in the rat portal vein model. Transmural field stimulation of the longitudinally mounted vein preparation evoked concomitant increases in the [ 3H]noradrenaline overflow and the integrated tension. Both responses were abolished by guanethidine or tetrodotoxin, whereas only the tension response was blocked by phentolamine. CGRP and VIP, both being present in intramural nerve fibers in the rat portal vein, were compared with atriopeptin II for modulatory effects. CGRP (100 nM) had no effect on the overflow of [ 3H]noradrenaline or the integrated tension response to transmural stimulation. VIP (30 nM) and atriopeptin II (30 nM) both caused significant reductions of both [ 3H]noradrenaline overflow and the integrated tension. These results indicate that the decreased tension response to transmural stimulation in the presence of VIP or AP II reflects the sum of both pre- and postsynaptic inhibitions.

Action of histamine on nerve mediated responses of the guinea-pig ileum.

The effect of histamine on the responses of the guinea-pig ileum to stimulation of intramural nerves and to some potential nonadrenergic, noncholinergic (NANC) neurotransmitters was analysed. During sustained tonic histamine contraction, electrical stimulation of all intramural nerves elicited a biphasic response (contraction followed by after-relaxation) and application of ATP and bradykinin caused relaxation of the ileum in contrast to their contractile effect on basal tension. Histamine reduced contractile and augmented relaxatory NANC responses, and prevented capsaicin from producing any contractile effect and from significantly influencing the NANC contractions. The present results suggest that, besides its direct effect, histamine activates intramural nerve fibres, mainly the sensory ones, and unmasks NANC relaxation thus modifying the mechanical activity.

Projections of peptide-containing neurons in rat small intestine

The distribution, origin and projections of nerve fibers containing vasoactive intestinal peptide, neuropeptide Y, somatostatin, substance P, enkephalin and calcitonin gene-related peptide were studied in the rat jejunum by immunocytochemistry and immunochemistry. Their origin was determined by the use of various procedures for extrinsic denervation (chemical sympathectomy, bilateral vagotomy or clamping of mesenterial nerves). The terminations of the different types of intramural nerve fibers were identified by examination of the loss of nerve fibers that followed local disruption of enteric nervous pathways (intestinal myectomy, transection or clamping). The majority of the peptide-containing nerve fibers in the gut wall were intramural in origin, each nerve fiber population having its own characteristic distribution and projection pattern. Nerve fibers emanating from the myenteric ganglia terminated within the myenteric ganglia and in the smooth muscle layers: those storing vasoactive intestinal peptide/neuropeptide Y, somatostatin and substance P were descending, those storing enkephalin were ascending and those containing calcitonin gene-related peptide projected in both directions. Nerve fibers emanating from the submucous ganglia terminated mainly within the submucous ganglia and in the mucosa: those storing calcitonin gene-related peptide or vasoactive intestinal peptide/neuropeptide Y were ascending and those storing substance P or somatostatin were both ascending and descending. Enkephalin nerve fibers could not be detected in the mucosa.

Attenuation of the responses to repeated cholinergic interventions in the isolated dog atrium.

In the isolated, blood-perfused canine right atrium, which was pretreated with propranolol, negative chronotropic and inotropic responses were evoked by stimulation of the intramural parasympathetic nerve fibers or by intra-arterial infusion of acetylcholine (ACh). Successive cholinergic interventions were applied; first, a conditioning intervention for 2 min was given, then this was followed by a test intervention for 4 min. The two interventions were separated by a rest period that varied from 15 to 240 s. The cardiac responses to the conditioning parasympathetic nerve stimulation quickly reached maximum levels, and then they "faded" or progressively diminished back toward the control level. The inotropic responses to the conditioning infusion of ACh (1 microgram/min) faded slightly but the chronotropic response did not. After the rest period, the test nerve stimulation evoked responses that also gradually faded with time. The maximal amplitude of the responses to the test stimuli were less than those to the conditioning stimuli. This reduction in the maximal amplitude of the cardiac responses to the test stimuli was more pronounced with high frequency stimulation (30 Hz) than with low frequency stimulation (5 Hz). The decrement was also more pronounced the shorter the rest period, and it was greater at earlier times after beginning the stimulation. Conversely, the maximal cardiac responses to test infusions of ACh were not appreciably less than the responses to the conditioning infusions. We conclude, therefore, that the diminution of the cardiac responses to the second test stimulation of the parasympathetic nerve fibers was mainly ascribable to a prejunctional rather than to a postjunctional mechanism.

Morphological studies of human gallbladder

I tried to make it clear three dimensional picture of the smooth muscle of the human gallbladder at first, and found a connection between the smooth muscle and the autonomic nerves in the wall. At autopsy the gallbladder without lesion was collected from 15 males and 7 females died of apoplexy, head injury or myocardial infarction. These material was fixed 10% neural formalin, impregnated into Masson Trichrome for smooth muscle, and modified Golgi method for nerve fivers, embeded in celloidin. I laid a picture after another after tracing serial tangential section of 8 micron by using camera lucida, and made three dimensional picture. In autonomic nerve in the wall three dimensional sections of 100-150 micron were made and observed with light microscope. Three dimensional muscle structure showed monounit structure, having meshwork structure from mucosal area to serous one. It was gratified the condition of the gallbladder contraction to illustrate a series of connection from mucosal aspect to serous one. Many fine nerve fivers were found forming plexuses in the subserous layer, muscular layer and mucosa. There was close relationship between smooth muscle and intramural nerve fibers, which especially distributed into meshwork structure of muscle. I could find morphological relation among the layers, such as three main plexuses. It was suggested that subserous plexus had extrinsic nature.